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JOURNAL OF INTERFERON & CYTOKINE RESEARCH 27:471–480 (2007)© Mary Ann Liebert, Inc.DOI: 10.1089/jir.2007.0178

Tumor Necrosis Factor-� Antagonism by the Murine Tumor Necrosis Factor-� Receptor 2-Fc Fusion

Protein Exacerbates Histoplasmosis in Mice

GEORGE S. DEEPE, JR.

ABSTRACT

Treatment of some inflammatory conditions with tumor necrosis factor-� (TNF-�) antagonists is efficacious,but such treatments are associated with infections with intracellular pathogens, including Histoplasma cap-sulatum. We explored protective immunity to H. capsulatum in mice given a fusion protein consisting of TNF-� receptor 2 (TNFR2) bound to the Fc portion of mouse IgG1. Intraperitoneal administration of this inhibi-tor exacerbated primary or secondary pulmonary infection at dosages ranging from 1 to 5 mg/kg. All micewith primary infection given the inhibitor succumbed to infection within 10–21 days of treatment. In sec-ondary histoplasmosis, mice receiving 1, but not 5, mg/kg survived treatment. Fungal burden was increasedeven if treatment with the inhibitor was initiated after the onset of infection. The inflammatory response ofthe lungs of mice given the inhibitor did not differ from that of mice given control vehicle. Susceptibility wasnot associated with major alterations in cytokines known to protect or exacerbate infection. However, ex-pression of nitric oxide synthase 2 (NOS2) was depressed early in primary infection. These results demon-strate that antagonism of endogenous TNF-� by this fusion protein modulates susceptibility. Impaired im-munity is not a result of altered cytokine responses or changes in the inflammation and may not bedemonstrable in other murine strains.

INTRODUCTION

IN RECENT YEARS, THERE HAS BEEN an enormous increase inthe number of biologic response modifiers (BRMs) available

for clinical use. These agents exert a host of biologic activitiesthat range from boosting immunity to dampening inflammation.Among those that are clinically available, the tumor necrosisfactor-� (TNF-�) antagonists have gained wide acceptance astreatment for a number of aggressive inflammatory diseases, in-cluding rheumatoid arthritis, ankylosing spondylitis, psoriaticarthritis, and Crohn’s disease.1 The expanding indications forthis class of therapeutic agents have increased the numbers ofpatients who are receiving this type of therapy. TNF-� not onlyis a key proinflammatory mediator, but it also is an importanthost defense molecule. This latter action is independent on theability of this cytokine to mobilize inflammatory cells.2 Thus,one of the consequences of TNF-� antagonism has been a surgein the infectious complications, especially intracellular patho-gens.3–5 Among these infectious complications has been thepathogenic fungus, Histoplasma capsulatum.4,5

Human infection with H. capsulatum occurs worldwide butis particularly prevalent in the southeastern and midwesternUnited States. Exposure to airborne mycelia and microconidiaeither produces no symptoms or may cause a mild influenza-like illness that spontaneously resolves. Once these fungal ele-ments have reached lung parenchyma, they transform intoyeasts, and this morphotype replicates within phagocytes untilcellular immunity is activated. Growth of the fungus is halted,but it can persist within tissues for many years in a dormantstate.6

The major mediators involved in protective immunity are Tcells and several cytokines. Interferon-� (IFN-�), granulocytemacrophage colony-stimulating factor (GM-CSF), and TNF-�are all necessary for the induction of a protective immune re-sponse to this fungus.7–12 Among these cytokines, TNF-� maybe the most important, based on studies in experimental ani-mals. Neutralization of it dramatically impairs protective im-munity in naïve mice and in those that possess preexistingimmunity.7 This effect is not observed for either IFN-� or GM-CSF. Although both IFN-� and GM-CSF are necessary for

Division of Infectious Diseases, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0560.

survival in primary histoplasmosis, their absence is not associ-ated with increased mortality in mice with secondary histo-plasmosis.8,11

Given the requirement of TNF-� for optimal host defensesagainst this fungus, we explored the influence of antagonismof this cytokine on the course of primary and secondary histo-plasmosis in mice. Although there is compelling evidence thatneutralization of endogenous murine TNF-� with either a poly-clonal or monoclonal antibody (mAb) exacerbates infec-tion,7,9–11 there are no data regarding the influence of anotherantagonist, a fusion protein consisting of TNF-� receptor 2(TNFR2) linked to the Fc portion of mouse IgG1.

MATERIALS AND METHODS

Mice

C57BL/6 mice were purchased from Jackson Laboratories(Bar Harbor, ME). Animals were housed in isolator cages andwere maintained by the Department of Laboratory Animal Med-icine, University of Cincinnati, which is accredited by the As-sociation for Assessment and Accreditation of Laboratory An-imal Care. All animal experiments were performed inaccordance with the Animal Welfare Act guidelines of the Na-tional Institutes of Health (NIH), and all protocols were ap-proved by the Institutional Animal Care and Use Committee ofthe University of Cincinnati.

Preparation of H. capsulatum and infection of mice

H. capsulatum yeast (strain G217B) was grown in HMMmedium for 60 h, washed, and enumerated.13 To produce in-fection in naïve mice, animals were inoculated intranasally (i.n.)with 2 � 106 H. capsulatum yeast cells in a 30-�L volume ofHBSS. Secondary infection was produced by infecting micewith 104 yeasts i.n and, 8 weeks later, infecting them with 2 �106 yeasts.

Organ culture for H. capsulatum

Recovery of H. capsulatum was performed as follows. Or-gans were homogenized in sterile saline and serially diluted.Homogenate (100 �L) was plated onto brain heart infusion agarplates containing 5% sheep blood and 5% glucose. Fungal bur-den was expressed as mean log10 colony-forming units (CFU)per whole organ � SEM. The limit of detection was 102 CFU.

Treatment of mice with murine TNFR2-Fc fusionprotein (p75-Fc) or with mAb to TNF-�

Mice were injected intraperitoneally (i.p.) with 1 mg/kg, 3mg/kg, or 5 mg/kg mouse murine p75 fused to the Fc portionof mouse IgG1 (provided by Dr. H. Kohno, Amgen Corpora-tion, Thousand Oaks, CA) dissolved in phosphate-bufferedsaline (PBS). Control animals were given an equal volume ofPBS. Rat antimouse TNF-� (from cell line XT-22.1) was pur-chased the National Cell Culture Center (Minneapolis, MN) andpurified. The cell line was obtained from Dr. J. Abrams (DNAX,Palo Alto, CA). Mice were injected i.p. with 1 mg mAb to TNF-�.

Isolation of lung leukocytes

Lung leukocytes were isolated as described.14 The ho-mogenates were filtered through a 60-�m nylon mesh (Spec-trum Laboratories Inc., Rancho Dominguez, CA) and washedthree times with HBSS. Leukocytes were isolated by separa-tion on a 40–70% Percoll (Pharmacia, Piscataway, NJ) gradi-ent. Cells were washed three times with HBSS prior to exam-ination.

Flow cytometry

The following antibodies were purchased from BD Bio-sciences (San Jose, CA): fluorescein isothiocyanate-conjugatedCD4 and CD8-FITC, phycoerythrin (PE)-conjugated Mac-3(macrophages), and PE-conjugated Gr-1 (neutrophils). Lungswere teased apart with the frosted ends of two glass slides andwashed with HBSS; 2 � 106 cells were incubated with 0.5 �gantibody in staining buffer (1% bovine serum albumin [BSA]in PBS) for 10 min at 4°C. The cells were washed in stainingbuffer, and fluorescence was measured using a FACScaliburflow cytometer (BD Pharmingen). The number of cells was cal-culated by multiplying the total number of leukocytes by thepercentage of cells bearing a specific surface receptor.

RNA isolation and cDNA synthesis

RNA was isolated from mouse lungs using trizol, and cDNAwas synthesized as previously described.14

Real-time polymerase chain reaction (qRT-PCR)

qRT-PCR for nitric oxide synthase 2 (NOS2) was performedusing the Taq-Man kit and primers from Applied Biosystems(Foster City, CA). Samples were analyzed on an ABI Prism7500 from Applied Biosystems. Transcript quantification wasstandardized to expression from an uninfected lung. In each ex-periment, the housekeeping gene hypoxanthine phosphoribosyltransferase was analyzed. The data were expressed in log10 ofrelative quantification. The conditions for amplification were50°C for 2 min, 95°C for 10 min, followed 40 cycles of 95°Cfor 15 sec and 60°C for 1 min.

Statistical analysis

ANOVA was used to compare groups. p � 0.05 was con-sidered statistically significant. Survival was analyzed using logrank.

RESULTS

Treatment of naïve mice with p75-Fc exacerbates infection

In our initial experiments, naïve mice were treated with 1,3, or 5 mg/kg of murine p75-Fc every other day beginning onthe day of infection. They were sacrificed on day 7 of infec-tion. Lungs and spleens were cultured for H. capsulatum. Asseen in Figure 1, the fungal burden was increased in each ofthe groups (p � 0.01). As each of the dosages caused indistin-guishable increases in the fungal burden, we assessed if thehighest and lowest dosage would impact survival of mice. Mice

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FIG. 1. Treatment with p75-Fc exacerbates primary histoplasmosis in mice. Groups of mice were infected with 2 � 106 yeastsi.n. and treated with p75-Fc or PBS on the day of infection and every 2 days thereafter. Mice were killed on day 7, and lungs(A) and spleens (B) were assessed for fungal burden. Results are expressed as means � SEM of 6–8 animals. (C) Survival curveof mice treated with 1 or 5 mg/kg of p75-Fc or PBS. (D) Mice (6–8 per group) were treated with p75-Fc or PBS every 4 days.Animals were followed for survival. One of two experiments is shown. **p � 0.01 vs. controls.

A B

C

D

were infected with 2 � 106 yeast cells and treated with p75-Fcor PBS every 2 days. Both dosages caused mice to succumb toinfection (Fig. 1C). Thus, TNF-� antagonism by this agent im-paired host defenses severely.

Administration of the fusion protein created for human useis less frequent than every 2 days and often is just once or twicea week. Therefore, we analyzed whether p75-Fc given to naïvemice twice weekly was associated with impaired immunity.Mice were infected with H. capsulatum and given 1, 3, or 5mg/kg of p75-Fc on the day of infection and every 4 days there-after. All mice administered the TNF-� antagonist succumbedto infection (Fig. 1D). Thus, decreasing the frequency of ad-ministration did not dramatically alter the outcome of infection.

Mice with preexisting immunity and p75-Fc

Residents of an endemic area may be repeatedly exposed toH. capsulatum and thus develop immunity. We sought to de-termine if p75-Fc altered the course of infection in mice withpreexisting immunity. Mice were exposed to 104 yeasts i.n., aninoculum that confers subsequent protection15; 8 weeks later,they were challenged with 2 � 106 yeasts and treated with p75-Fc or given PBS, and survival was followed. Mice given 1mg/kg of p75-Fc every 2 days survived infection, whereas thosegiven 3 mg/kg or 5 mg/kg every 2 or 4 days did not (Fig. 2A).To determine if the failure to survive was a result of an increasein CFU, we analyzed fungal burden in mice that received 3mg/kg or 5 mg/kg every 4 days. In those mice, the fungal bur-den was increased compared with controls (Fig. 2B,C).

Does p75-Fc impact fungal burden if initiated during infection?

Naïve and immune mice were infected with H. capsulatumyeasts, and 5 mg/kg of p75-Fc was begun on the day of infec-tion or on day 2 or 4 after infection. Mice were treated every2 days i.p. with this antagonist or with PBS. The fungal bur-den in lungs and spleens was elevated in each of the groups re-ceiving p75-Fc and significantly exceeded (p � 0.01, p � 0.05,spleens) that of controls (Fig. 3A,B). There were no significantdifferences (p � 0.05) among the groups of treated mice. Insecondary histoplasmosis, treatment initiated after infectionalso blunted the protective immune response (Fig. 3C,D).

Inflammatory response in mice given p75-Fc

Because administration of p75-Fc altered the protective im-mune response to H. capsulatum, we queried if it modulatedthe nature of the inflammatory response to this fungus. Naïveand immune mice were infected with 2 � 106 yeasts and given5 mg/kg every 4 days. We chose this regimen because it wasthe most potent dosage to dampen the protective immune re-sponse in both primary and secondary infection. Lungs wereremoved on days 3 and 7 of infection, and lung leukocytes wereisolated. We analyzed the number of macrophages (Mac-3�),neutrophils (Gr-1�), and CD4� and CD8� cells, as these arethe principal regulators of immunity to this fungus. At day 7 ofinfection, the numbers of macrophages and neutrophils were in-creased (p � 0.05) in animals receiving p75-Fc (Fig. 4A,B). Insecondary infection, the numbers of each population were sim-ilar at days 3 and 7 of infection (Fig. 4C,D).

Cytokine response

The lungs of mice were analyzed for alterations in media-tors that are known to regulate the host response to this fungus.We analyzed by qRT-PCR transcription of interleukin-4 (IL-4)and IL-10, which inhibit protective immunity, IFN-�, whichpromotes protective immunity, and NOS2, which induces thesynthesis of NO, a key mediator of protection. In primary in-fection, there were no differences in transcription of IL-4, IL-10, or IFN-� at day 3 or 7 (Fig. 5). However, there was a mod-est decline in NOS2 at day 3, but by day 7, this value wassimilar to that of controls. In secondary infection, the resultsfor the cytokines and NOS2 were similar (Fig. 5).

Is effect of p75-Fc similar to that of mAb to TNF-�?

As mentioned in the Introduction, much of the literature onthe influence of TNF-� and H. capsulatum has been derivedfrom studies using polyclonal antibody or an mAb. We infectednaïve mice with 2 � 106 yeasts and treated them with a singledose of 1 mg/kg or 5 mg/kg of p75-Fc every 4 days or with 1mg of anti-TNF-�. Mice were killed at day 7 of infection andassessed for fungal burden in lungs and spleens. CFUs in lungsof mice given either p75-Fc or mAb to TNF-� were elevated(p � 0.01) compared with controls (Fig. 6). Analysis of thetreated groups showed that CFUs in lungs of mice given 1mg/kg but not 5 mg/kg of p75-Fc were modestly less (�0.7log10 CFU, p � 0.05) than those of mAb recipients.

We conducted the same experiment in immune mice, withthe exception that we used only 5 mg/kg every 4 days, as the1 mg/kg dose did not impact survival. As shown in Figure 6,the effect of the mAb was far more pronounced when comparedwith p75-Fc (p � 0.01).

DISCUSSION

A number of risk factors for progressive disseminated histo-plasmosis have been identified from clinical studies.16 Theseinclude but are not limited to AIDS, immunosuppressive med-ications, and age. Among the many immunosuppressive agentsavailable, one of the current classes of agents that are knownto enhance susceptibility to disseminated histoplasmosis is theTNF-� antagonists.4,5 A recent report suggests that treatmentwith infliximab is associated with a higher risk of developinghistoplasmosis than is treatment with etanercept.5 The reasonsfor these differences remain unknown. In this study, we en-deavored to determine if the protein consisting of the TNFR2receptor fused with IgG1 Fc molecule exacerbated infectionwith H. capsulatum in mice.

Using several different dosages, we found that naïve micemanifested increased CFU and did not survive this treatment.The increased susceptibility to infection was noted if p75-Fcwas administered every other day or every 4 days. The enhancedsusceptibility of mice given p75-Fc was not associated withmarked decrements in the inflammatory cells in the lungs. Infact, two effector cell populations, macrophages and neu-trophils, were increased in recipients of p75-Fc. The latter areknown to inhibit the intracellular growth of H. capsulatum.17,18

Despite this effect, the mice still succumbed to infection.The increased number of macrophages present in lungs of

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mice with primary infection might have added to the aggres-sive nature of the infection, as this cell population often servesas an intracellular haven for the fungus.19–21 Unless the cellsare activated, the organism can replicate unimpeded within thispopulation of phagocytes. IFN-� is known to activate peritonealmacrophages but does not enhance the anti-Histoplasma activ-ity of lung macrophages.7,22 Colony-stimulating factors (CSF)

are known to augment short-term (within 2 h) killing of H. cap-sulatum within alveolar macrophages in vitro, but their effecton fungicidal activity in vivo is unknown.23 Antagonism ofTNF-� activity in vivo by mAb is not known to depress GM-CSF levels, and thus it seems unlikely that p75-Fc would mod-ulate the production of this family of cytokines.

In secondary infection, susceptibility to infection was clearly

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FIG. 2. Effect of p75-Fc on secondary histoplasmosis. Mice were infected with 104 yeasts i.n. and 8 weeks later challengedwith 2 � 106 yeasts i.n. and given 1 or 5 mg/kg of p75-Fc every 2 days. (A) Survival was assessed. (B and C) Mice were treatedwith 5 mg/kg of p75-Fc every 2 days, and fungal burden was assessed in lungs (B) or spleens (C) at day 7 of infection. Resultsrepresent mean � SEM of 6–8 per group. **p � 0.01.

dependent on the dosage of p75-Fc. This distinction was notobserved in naïve mice, although those that received a lowerdose did survive longer. Administration of 1 mg/kg of p75-Fcevery 2 days did not impact survival of immune mice, whereasthe higher dosage did whether it was administered every 2 daysor every 4 days. These results signify that preexisting immu-nity may modulate susceptibility to the severity of infection.Moreover, administration of p75-Fc to immune mice did notcause a modulation of either the inflammatory response or the

cytokines that were analyzed. This finding is in contrast to theresults with the mAb to TNF-�, where IL-4 and IL-10 are bothelevated following neutralization in vivo in secondary histo-plasmosis.7

In experimental murine histoplasmosis, the first week of in-fection is the crucial time for the subsequent development ofimmunity.14 During this time frame, such mediators as IL-12,IFN-�, GM-CSF, and TNF-� are synthesized, and their releaseis necessary for protective immunity.7,9,14,15,24 TNF-� appears

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FIG. 3. p75-Fc alters fungal burden when given in active infection. Naïve mice (A and B) or immune mice (C and D) wereinfected with 2 � 106 yeasts i.n. and treated on the day of infection or on day 2 or 4 after infection. The biological was admin-istered every 2 days thereafter. Results represent mean � SEM of 6 mice. *p � 0.05 vs. controls; **p � 0.01 vs. controls.

to be one of the earliest cytokines synthesized, followed by en-gagement of the IL-12-IFN-� axis and GM-CSF.9 Impairmentof any of those mediators disrupts immunity. Persistent pro-duction of TNF-� has been shown to be important in the evo-lution of the protective immune response.24 Neutralization ofthis cytokine within the first 5 days of this infection blunts theability of mice to clear infection. Likewise, we determined thatadministration of p75-Fc in established primary or secondaryinfection exacerbated infection even when it was initiated 4days into infection. These results support our assertion that per-sistent production of TNF-� is critical for the optimal expres-sion of protective immunity.

Analysis of the cytokine response by mice given the inhibi-tor demonstrated that none of the cytokines assayed was mod-ulated. Only NOS2, which leads to NO production, was mod-ulated in primary infection. These results are similar to thefindings for mice given mAb to TNF-�. Animals receiving thismAb manifested a marked decrease in NO release; however,they differ from TNFR2�/� mice, in which IFN-� is de-pressed.11,12 As the circulating antagonist binds TNF-�. thusremoving it from engaging TNFR, the host response appears tobe similar to that of TNF-�-neutralized mice. Although we mea-sured NOS2 transcripiton, we have found that it correlates wellwith NO release.25 In secondary infection, no alterations were

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FIG. 4. Inflammatory cells in lungs of mice administered p75-Fc. Mice were treated with 5 mg/kg p75-Fc every 4 days be-ginning on the day of infection. Lung leukocytes (�95% CD45�) were analyzed on (A and C) day 3 and (B and D) day 7 forseveral cell subpopulations. Results represent mean � SEM of 4–8 animals. *p � 0.05.

FIG. 5. qRT-PCR of cytokines and NOS2 for primary and secondary histoplasmosis. RNA from lungs of mice was prepared,and cDNA was synthesized. qRT-PCR was performed on lungs using commercially available primers. Expression in primary in-fection was compared with uninfected lungs and in secondary infection with lungs from mice that had received 104 yeasts 8weeks before rechallenge. Results represent mean � SEM of 4–6 animals per group. *p � 0.05.

observed in IL-4 or IL-10 in the lungs of mice given p75-Fc.In contrast, the lungs of mice given mAb to TNF-� exhibitedincreased IL-4 and IL-10 in response to rechallenge with H.capsulatum. These disparate results indicate that the effects ofbiologicals can vary even though their net effect is similar. Theprecise mechanisms by which p75-Fc modulates immunity re-main to be determined; it appears that cytokine modulation isnot one of them. Recently, it was shown that T cells from micedeficient in TNF-� are dysfunctional in their ability to conferprotection, and this effect is independent of their IFN-� syn-thesis capacity.24 Thus, it is possible that the effect of p75-Fcis directed at the T cell compartment independent of modulat-ing IFN-� levels.

Additional studies found that the effect of p75-Fc on fungalburden in primary infection was similar to that of an mAb to

TNF-�. On the other hand, there was a difference in the lungsof mice with secondary infection. Although the precise reasonfor this difference is not known, several possibilities may ex-plain it. One of the simplest explanations is the disparity in pro-tein content, but this seems unlikely given that the two com-pounds did not produce different results in primary infection.Another possibility is that the metabolism, the pharmacologicdistribution, absorption, or the biologic half-life of the tworeagents may differ in secondary but not primary infection. Vir-tually nothing is known about these parameters with either themAb or the murine p75-Fc. A better understanding of the phar-macology of these reagents will likely improve our under-standing of the differences in the impact of these agents.

In summary, p75-Fc reduced the ability of mice to surviveinfection with H. capsulatum. This effect was observed in both

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FIG. 6. Comparison of p75-Fc and mAb to TNF-�. Mice were infected with H. capsulatum and given p75-Fc every 4 days or1 mg mAb to TNF-� at the time of infection. On day 7, mice were killed, and CFUs were determined in lungs (A and C) andspleens (B and D) of mice with primary (A and B) or secondary (C and D) histoplasmosis. Results represent mean � SEM of4–8 animals. *p � 0.05; **p � 0.01.

primary and secondary infection, thus supporting the impor-tance of TNF-� in regulating immunity to both phases of in-fection. However, mice that had been exposed to the fungus didmanifest a dose-dependent difference in susceptibility to thep75-Fc. The mechanisms by which this compound and mAb toTNF-� dampen immunity require further exploration.

ACKNOWLEDGMENTS

This work was supported by a grant from Amgen Corpora-tion and by Public Health Service grants AI-42747 and AI-34361 and a Merit Review from the Veterans Affairs. I thankMichael McGuinness for excellent technical assistance. Dr.Deepe has been a consultant for Amgen Corporation and hasreceived a grant from Amgen Corporation.

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Address reprint requests or correspondence to:Dr. George S. Deepe, Jr.

Division of Infectious DiseasesUniversity of Cincinnati College of Medicine

Cincinnati, OH 45267-0560

Tel: (513) 558-4704Fax: (513) 558-2089

E-mail: george.deepe@uc.edu

Received 12 December 2006/Accepted 3 January 2007

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